Labelling of nucleic acids and/or oligonucleotides, with reporter molecules is important in many areas of chemical and biological research. Due to the fact that single stranded oligonucleotides hybridize with complementary single or double stranded oligonucleotides, labelled oligonucleotides can be used as probes in cloning procedures, blotting procedures such as Northern blot analysis, and in situ hybridization procedures. Additionally, labelled oligonucleotides can be used in conjunction with oligonucleotide amplification procedures such as the Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA), and Ligase Chain Reaction (LCR) to detect the presence of amplified oligonucleotides. Thus, labelled oligonucleotides are used for both qualitative and quantitative analyses of target nucleic acid molecules.
Oligonucleotides can be labelled with several different types of reporter molecules. For example, oligonucleotides can be labelled with radioisotopes such as .sup.32 P, .sup.3 H, .sup.14 C, .sup.35 S, .sup.125 I, or .sup.131 I. Oligonucleotides may also be labelled with non-isotopic labels such as fluorescein, biotin, digoxigenin, and alkaline phosphatase. However, when using labelled oligonucleotides for the identification and quantification of target nucleic acids, fluorescent labels have been favored as they provide a sensitive, non-radioactive means for the detection of probe hybridization. The fluorescent labels may be used alone, or in conjunction with quenching dyes in fluorescence energy transfer reactions. Fluorescence energy transfer occurs between a donor fluorophore and an acceptor or quenching dye when the absorption spectrum of the acceptor dye overlaps the emission spectrum of the donor fluorophore, and the two dyes are in close proximity. Upon excitation of the donor molecule, for example with ultraviolet energy, the energy emitted from the donor molecule is transferred to the neighboring acceptor molecule which accepts and quenches this energy. This acceptance of the energy by the acceptor results in quenching of donor fluorescence. The overall effect of such energy transfer is that the emission of the donor is not detected until the donor and acceptor are separated, for example upon hybridization of a labeled probe to a target nucleotide.
In practice, the donor and acceptor molecules may either reside on complementary oligonucleotides or on a single oligonucleotide. When incorporated into complementary oligonucleotides, quenching occurs upon hybridization of the separately labelled oligonucleotides. In contrast, when the donor and acceptor are linked to a single oligonucleotide, hybridization to the target oligonucleotide usually results in reduced quenching due to an increased distance between the donor and acceptor which decreases the effect of energy transfer. Reduced quenching is observed as increased ability to detect the energy emitted from the donor. For example, an acceptor and donor may be linked to the ends of a self-complementary oligonucleotide such that under non-hybridizing conditions a hairpin is formed which brings the acceptor and donor into close proximity and causes quenching. Hybridization of the self-complementary oligonucleotide results in linearization of the hairpin and reduced quenching. Additionally, to further contribute to the change in fluorescence upon hybridization, a restriction endonuclease site may be placed between the acceptor and donor dyes such that the site is only cleavable in the presence of target binding.
Two of the quenching or acceptor molecules that are used with the detection techniques described above are DABSYL and DABCYL. The term "DABSYL" refers to a 4-dimethylaminophenylazo-benzenesulfonyl acid while the term "DABCYL" refers to a 4-dimethylaminophenylazo-benzene-4'-carbonyl structure. The term "DABCYL thioamide" refers to a 4-dimethylaminophenylazophenyl-4'-isothiocyanate. These molecules have absorption spectra that overlap the emission spectrum of donor molecules such as EDANS, BODIPY dyes, fluorescein, tetramethylrhodamine, Texas Red, rhodamine-X, Cy3 and Cy5. DABCYL, DABSYL and DABCYL thioamide are non-fluorescent chromophores, and therefore provide an advantage over other quenchers since they do not fluoresce when exposed to emission from the donor molecule or to the excitation wavelengths used to excite the donor.
Several reagents have been produced for linking acceptor dyes, such as DABCYL, to oligonucleotides. For example, Glen Research makes a 3'-DABCYL CPG 500 product that allows linkage of the DABCYL molecule to the 3' end of an oligonucleotide (1998 Catalog, Glen Research, Sterling, Va.). One drawback to this product however, is that the DABCYL molecule cannot be linked to the interior of the oligonucleotide chain nor to the 5' end of the oligonucleotide chain. Another product, DABCYL-cdT, facilitates linkage of the DABCYL molecule to the internal nucleotides of a growing oligonucleotide chain, but is limited in application to the nucleotides thymidine and uridine (Glen Research).
Derivatives of DABSYL-type reactive dyes present problems in post-labeling of oligonucleotides due to their high hydrophobicity and low aqueous solubility. These characteristics require the coupling reaction with 5'-amino-linked DNA or RNA to be performed in buffers with a high percentage of dimethylsulfoxide (DMSO) or dimethylformamide (DMF) and complicates purification of labeled DNA or RNA from dye by-products.
Accordingly, what is needed are labelled compositions that may be used as reagents during oligonucleotide synthesis for the linkage of labels, such as acceptor dyes, to the 5' end of oligonucleotides. Also needed are labelled compositions, capable of linking to the 5' end of oligonucleotides, which are less hydrophobic than labelled compositions taught in the prior art. What is also needed are methods for labelling oligonucleotides that reduce or eliminate the problems inherent in prior art methods such as the use of hydrophobic solvents and hydrophobic buffers that complicate purification of the labelled oligonucleotides.